Tyrosine phosphorylation of proteins is involved in an increasing number of cellular signalling events. It was originally implicated in signalling by paracrine- or autocrine-acting growth factors, and endocrine hormones such as insulin (see Yarden, Y. et al., Annu. Rev. Biochem. 57:443–478 (1988) for review). It is now clear that this posttranslational modification is also involved in diverse processes such as the activation of cells of the immune system by antigens (Klausner, R. D. et al., Cell 64:875–878), signalling by lymphokines (Hatakeyama, M. et al., 1991 Science 252:1523–1528 (1991); Mills, G. B. et al., J. Biol. Chem. 265:3561–3567 (1990)), and cellular differentiation and survival (Fu, X.-Y. 1992 Cell 70:323–335; Schlessinger, J. et al. 1992 Neuron 9:1–20; Velazquez, L. et al., 1992 Cell 70:313–322). In view of the diversity of processes in which tyrosine phosphorylation is involved, it is not surprising that links are also emerging with the process of cell adhesion and cell-cell contact.
The identification of several growth factor receptors and retroviral oncogenes as tyrosine-specific protein kinases indicated that protein phosphorylation on tyrosine residues plays a key role in cellular growth control. This notion has recently received support by the observation that the level of tyrosine phosphorylation of enzymes thought to play an important role in signal transduction (such as phospholipase C) correlates with their increased activity upon growth factor stimulation, thus establishing a functional role for tyrosine phosphorylation (Ullrich, A., et al., Cell 61:203–212 (1990)).
Most of the processes in which tyrosine phosphorylation is implicated involve the transduction of a signal through the cell membrane. In its best understood fashion, this can occur through dimerization-mediated activation of members of the receptor tyrosine kinase family by soluble ligands (reviewed in Ullrich, A. et al. 1990 Cell 61:203–212). However, modulation of receptor tyrosine kinase activity can also occur by membrane-bound ligands on neighboring cells, as in the case of the interaction between the sevenless kinase and the bride of sevenless protein (Rubin, G. M. 1991, Trends in Genetics 7:372–376). Recently, receptor-like tyrosine kinases were described with an extracellular domain similar to that of cell adhesion molecules of the CAM-family (e.g. Axl and Ark (O'Bryan, J. P. et al., 1991 Mol. Cell. Biol. 11:5016–5031; Rescigno, J. et al., 1991 Oncogene 6:1909–1913)). Such observations may implicate tyrosine phosphorylation as a more broadly used direct downstream effector mechanism for precise cell-cell recognition and signalling events. Members of the non-receptor family of tyrosine kinases have also in several instances been shown to be associated with other proteins with a trans-membrane topology, examples being the association of the Lck and Fyn kinases with the CD4 protein and T-cell receptor complex components respectively (Haughn, L. et al., 1992 Nature 358:328–331; Samelson, L. E. et al., 1992 Proc. Natl. Acad. Sci. USA 87:4358–4362; Veillette, A. et al., 1988 Cell 55:301–308). However, the mechanism by which kinase activity is modulated in these instances is not understood.
The degree and pattern of phosphorylation of tyrosine residues on cellular proteins are regulated by the opposing activities of protein-tyrosine kinases (PTKases; ATP:protein-tyrosine O-phosphotransferase, EC 2.7.1.112) and protein-tyrosine-phosphatases (PTPases; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). The structural characteristics and evolution of PTKases as well as their role in the regulation of cell growth have been reviewed (Hunter, T., et al., Annu. Rev. Biochem. 54:897–930 (1985); Ullrich, A., et al., supra).
2.1. PTKases
Tyrosine kinases comprise a discrete family of enzymes having common ancestry with, but major differences from, serine/threonine-specific protein kinases (Hanks, S. K. et al., (1988) Science 241:42–52). The mechanisms leading to changes in activity of tyrosine kinases are best understood for receptor-type tyrosine kinases which have a transmembrane topology (Ullrich, A. et al., supra). With such kinases, the binding of specific ligands to the extracellular domain of these enzymes is thought to induce their oligomerization leading to an increase in tyrosine kinase activity and activation of the signal transduction pathways (Ullrich, A. et al., supra). The importance of this activity is supported by the knowledge that dysregulation of kinase activity through mutation or over-expression is a mechanism for oncogenic transformation (Hunter, T. et al., supra; Ullrich, A. et al., 1990, supra).
2.2. PTPases
The protein phosphatases are composed of at least two separate and distinct families (Hunter, T. Cell, 58:1013–1016 (1989)), the protein serine/threonine phosphatases and the protein tyrosine phosphatases. This is in contrast to protein kinases, which show clear sequence similarity between serine/threonine-specific and tyrosine-specific enzymes.
There appear to be two basic types of PTPase molecules. The first group is comprised of small, soluble enzymes that contain a single conserved phosphatase catalytic domain, and include (1) placental PTPase 1B (Charbonneau, H. et al., Proc. Natl. Acad. Sci. 86:5252–5256 (1989); Chernoff, J. et al., Proc. Natl. Acad. Sci. USA 87:2735–2789 (1990)), (2) T-cell PTPase (Cool, D. E. et al., Proc. Natl. Acad. Sci. USA 86:5257–5261 (1989)), and (3) rat brain PTPase (Guan, K., et al., Proc. Natl. Acad. Sci. USA, 87:1501–1505 (1990).
The identification of a tyrosine phosphatase homology domain has raised new interest in the potential of PTPases to act as modulators of tyrosine phosphorylation (Kaplan, R. et al. 1990 Proc. Natl. Acad. Sci. USA 87:7000–7004; Krueger, N. X. et al., 1990 EMBO J. 9:3241–3252; see, for review, Fischer, E. H. et al., 1991 Science 253:401–406).
The second group of PTPases is made up of the more complex, receptor-linked PTPases, termed R-PTPases or RPTPs, which are of high molecular weight and contain two tandemly repeated conserved domains separated by 56–57 amino acids. RPTPs may be further subdivided into four types based on structural motifs within their extracellular segments.
One example of RPTPs are the leukocyte common antigens (LCA) (Ralph, S. J., EMBO J., 6:1251–1257 (1987); Charbonneau, H., et al., Proc. Natl. Acad. Sci. USA, 85:7182–7186 (1988)). LCA, also known as CD45, T200 and Ly-5 (reviewed in Thomas, M. L., Ann. Rev. Immunol. 7:339–369 (1989)) comprises a group of membrane glycoproteins expressed exclusively in hemopoietic (except late erythroid) cells, derived from a common gene by alternative splicing events involving the amino terminus of the proteins.
Other examples of RPTPs are the LCA-related protein, LAR (Streuli, M. et al., J. Exp. Med., 168:1523–1530 (1988)), and the LAR-related Drosophila proteins DLAR and DPTP (Streuli, M., et al., Proc. Natl. Acad. Sci. USA, 86:8698–8702 (1989)). Jirik et al. screened a cDNA library derived from the human hepatoblastoma cell line, HepG2, with a probe encoding the two PTPase domains of LCA (FASEB J. 4:A2082 (1990), abstr. 2253) and discovered a cDNA clone encoding a new RPTP, named He-PTP. The HePTP gene appeared to be expressed in a variety of human and murine cell lines and tissues.
A large number of members of the RPTP family, called type II RPTPs, display an extracellular domain containing a combination of Ig-domains and fibronectin type III repeats (Fn-III), features typically encountered in cell adhesion molecules (CAMs) (Gebbink, M. F. B. G. et al., 1991 FEBS Lett; 290:123–130; Streuli, M. et al., 1988 J. Exp. Med. 168: 1523–1530). An analysis of the expression patten of several R-PTPases in the developing Drosophila CNS suggests some function of these molecules in aspects of axon guidance and outgrowth (Tian, S. S. et al., 1991 Cell 67:675–685; Yang, X. et al., 1991. Cell 67:661–673), an observation winch might be related to the ability of R-PTPases to control the activity of src-family tyrosine kinases (Mustelin, T. et al., 1989 Proc. Natl. Acad. Sci. USA 86:6302–6306; Ostergaard, H. L. et al., 1989 Proc. Natl. Acad. Sci. USA 86:8959–8963; Zheng, X. M. et al., 1992 Nature 359:336–339). Other studies have raised the possibility that certain R-PTPases may function as tumor suppressor genes, e.g. by controlling contact inhibition (LaForgia, S. et al., 1991 Proc. Natl. Acad. Sci. USA 88:5036–5040). Elevation of cellular phosphotyrosine may occur through mechanisms other than the activation of a tyrosine kinase itself. For instance, expression of the v-crk oncogene, though not a tyrosine kinase, induces the phosphorylation of tyrosine residues through a poorly understood mechanism (Mayer, B. J. et al. (1988) Nature 332, 272–275). Potentially, such an outcome could result from either mutation of the substrate or through a general decrease in cellular phosphatase activity, especially in view of the normally high turnover rate of cellular tyrosine-phosphate (Sefton, B. M. et al. (1980) Cell 20:807–816). The latter possibility is suggested by the demonstration that tyrosine phosphatase inhibitors can “reversibly transform” cells (Klarlund, J. K. Cell 41: 707–717 (1985)). PTPases could therefor act as recessive oncogenes.
While we are beginning to understand more about the structure and diversity of the PTPases, much remains to be learned about their cellular functions. Thus, a better understanding of, and an ability to control, phosphotyrosine metabolism, requires knowledge not only the role of PTKase activity, but the action of PTPase enzymes as well. It is clear in the art that further delineation of structure-function relationships among these PTPases and RPTP membrane receptors are needed to gain important understanding of the mechanisms of cell growth, differentiation, and oncogenesis.